ABSTRACT: General anesthesia is essential for modern procedural medicine. There is a compelling need to find superior alternatives to current sedative-hypnotics, which derive from a few drug classes identified before 1980, and also cause undesirable or harmful side effects, especially in vulnerable populations. To date, strategies to discover new anesthetics are based on molecular models, mostly GABAA receptors, that are targets for a few potent intravenous hypnotics. Our long-term goals are to identify new sedative-hypnotic chemotypes that may improve patient care, while also advancing molecular and neurobiological research on anesthetic mechanisms. We hypothesize that a wide variety of undiscovered potent sedative-hypnotic chemotypes exists. These may modulate GABAA receptors through several known sites, or act through other known or perhaps unknown mechanisms. Our novel strategy uses concurrent video motion analysis of up to 96 zebrafish larvae as a high-throughput un-biased stimulus-response test platform to accelerate discovery and characterization of new potent sedative-hypnotics. Zebrafish larvae immersed in solutions of non-volatile drugs rapidly establishes steady-state conditions for assessing pharmacodynamic effects. Combining this approach with new transgenic zebrafish lines harboring mutations that alter or eliminate known and suspected general anesthetic targets in the nervous system will also accelerate exploration of molecular and neurobiological mechanisms. In Aim 1, we will use video analysis tools to assess both baseline motion and photomotor response probability in up to 96 zebrafish larvae simultaneously. We have developed robust experimental and data analysis approaches to both screen for new sedative-hypnotics and determine sedative and hypnotic potencies (Aim 1a). We already have identified several potent active lead compounds in a drug library from the Boston University Center for Medical Discovery (BUCMD). New active leads will also be tested for activity/potency in both tadpoles and rats (Aim 1b). Collaborators at BUCMD will also synthesize structural variants of selected lead compounds, enabling exploration of structure-activity relationships for sedation and hypnosis in zebrafish, as illustrated in preliminary data (Aim 1c). In Aim 2, we will explore the molecular mechanisms of new sedative-hypnotics using voltage-clamp electrophysiology in a panel of heterologously expressed ion channels (GABAARs, GlyRs, NMDA-Rs, neuronal nAChRs, HCN1 and TREK-3) or antipamezole antagonism of ?2 adrenergic receptors (Aim 2a). Sedative-hypnotics that potentiate GABAARs, will also be tested in a series of receptor mutants to assess selectivity for known subsites (Aim 2b). In Aim 3, we will use CRISPR-Cas9 to create transgenic zebrafish lines for testing the roles of drug targets in sedation and hypnosis. We will first create a panel of GABAAR subunit knock-out and knock-in lines (Aim 3a; ?3 and ? KOs are made). We will also make knockouts for HCN1 (already made), TREK-3, K2P and ?2 AdRs (Aim 3b). Sedative and hypnotic potencies of known and novel drugs will be compared in mutant vs. wild-type zebrafish.